Ion channels and receptors are membrane interface macromolecules that mediate key physiological and pathological activity. Understanding their three-dimensional structure-activity relationship has been a central and yet elusive goal of biophysicists. The nanoscale size and structural complexity, such as an ion channel's subunit architecture and the central permeable pore, their membrane insertion and lipid-protein interactions put major constraints on their structural analysis. High resolution 3D structure of ion channels is being examined primarily with X-ray diffraction and electron microscopy. Ion channel activity is commonly analyzed by patch clamping and fluorescence microscopy. However, integration of these techniques into a combined system for the direct structure-function activity of ion channels is limited. Atomic force microscopy (AFM) provides high resolution structural information for many biological macromolecules, including ion channels and receptors. AFM images surfaces, the primary structural domains where ligands and other gating agents interact and alter channel activity. AFM allows online addition of pharmacologic or (patho-) physiologic stimuli. Its open architecture allows integration with other techniques permitting imaging of channel-stimuli complex, resulting 3D conformations and channel properties such as permeability, conductance, energetics, and mechanics. The main focus of this application is two-fold: a) to have a practical integrated double chamber AFM with the Support Chip, combine them with high sensitive electrical and fluorescence measuring tools and b) to use them to study hypothesis-driven mechanistic questions about the structure-activity relation for two different channels: i) Connexin hemichannels for high resolution and flexibility work, especially its cytoplasmic face, for which very little is known but much is speculated and for molecular permeability and ii) KcsA channels that form nice, high-conductance pH gated channels. Hemichannel will be used for structural and permeability study. KcsA channel will be used to test simultaneous measurement of conductance, the channel surface topography, and the opening of the bundle crossing implicated in channel opening and closing. Specific Aims of the application are: Aim 1: Design Support Chip with single or multiple nanopore(s) and integrate it with a high resolution AFM. Aim 2: Image 3D topography of reconstituted hemichannels and full-length or truncated KcsA channels. Aim 3: Examine the role of channel subunit structural changes in the open/closed functional states. This includes high resolution imaging of the structure while simultaneously measuring permeability, of dyes and large molecules specific to hemichannels, with integrated TIRF microscopy and examining role of various known gating agents, site-directed peptides and antibodies on the permeability. For KcsA channels, both truncated as well as full- length channels will be studied - truncated channel for 3D structure imaging and the full-length channel for imaging bundle crossing to correlate channel gating. While Aims 1 and 3 are interdependent to some extent, Aim 2 is independent and only relies on designing sharp AFM tips with low spring constant and high S/N ratio. PUBLIC HEALTH RELEVANCE: Ion channels and receptors are membrane structures that determine key physiological functions. Abnormality in their structure and activity underlie major human diseases, yet there is a limited understanding of their structure-activity relation. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D structure, yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying human diseases. The long term significance of our study is considerable. The techniques developed in our study, namely combining an equivalent of EM and single channel conductance (patch clamping) and permeability capability will provide a major breakthrough for 3D structure-function study of ion channels and receptors. Our results from study of hemichannels and potassium channel will provide new paradigms to examine the mechanism of cell-extracellular exchanges and their role in modulating tissue homeostasis. Mechanistic information gained from our study will then be used for designing effective prevention and treatment of diseases, including cardiac arrhythmias, neurodegenerative diseases, and cancer.